การประชุมวิชาการเรื่อง Analytical Method Validation for FDA Compliance

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 47

Example : Intermediate precision

Conditions: different analyst, different HPLC, different day

Sample No. %Assay

Analyst 1, HPLC1 Analyst 2, HPLC 2

1 101.5 100.7
2
3 101.7 100.5
4
5 101.9 101.0
6
Average 102.1 100.7
RSD (%)
RSD (%) overall 101.5 100.6

101.6 100.1

101.7 100.6

0.2 0.3

0.6

Acceptance criteria: RSD : not more than 2.0 %

55

Detection limit vs Quantitation
limit

‘Know that it’s there’
vs

‘Know how much is there’

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Detection limit

(means)

Is any of it present?

Is it there?

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Quantitation limit

How much of it is present???

How much of it is there?

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 49

DETECTION LIMIT

• The detection limit of an individual analytical
procedure is the lowest amount of analyte in
a sample which can be detected but not
necessarily quantitated as an exact value

• Several approaches for determining the detection
limit are possible, depending on whether the
procedure is a non-instrumental or instrumental.

59

Based on Visual Evaluation

• Visual evaluation may be used for non-
instrumental methods but may also be used with
instrumental methods.

• The detection limit is determined by the analysis
of samples with known concentrations of analyte
and by establishing the minimum level at which
the analyte can be reliably detected .

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Based on Signal-to-Noise

• This approach can only be applied to analytical
procedures which exhibit baseline noise.

• Determination of the signal-to-noise ratio is performed by
comparing measured signals from samples with known
low concentrations of analyte with those of blank
samples and establishing the minimum concentration at
which the analyte can be reliably detected.

• A signal-to-noise ratio between 3:1 or 2:1 is generally
considered acceptable for estimating the detection limit.

61

Based on the Standard Deviation of the
Response and the Slope

The detection limit (DL) may be expressed as:
DL = 3.3 /S
where  = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration
curve of the analyte.

62

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 51

Estimate of 

• Based on the Standard Deviation of the Blank

– Measurement of the magnitude of analytical background
response is performed by analyzing an appropriate number of
blank samples and calculating the standard deviation of these
responses

• Based on the Calibration Curve

– A specific calibration curve should be studied using samples
containing an analyte in the range of DL.

– The residual standard deviation of a regression line or the
standard deviation of y-intercepts of regression lines may be
used as the standard deviation.

63

Recommended Data

• The detection limit and the method used for
determining the detection limit should be presented.

• If DL is determined based on visual evaluation or based
on signal to noise ratio, the presentation of the relevant
chromatograms is considered acceptable for
justification.

• In cases where an estimated value for the detection limit
is obtained by calculation or extrapolation, this estimate
may subsequently be validated by the independent
analysis of a suitable number of samples known to be
near or prepared at the detection limit

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QUANTITATION LIMIT

• The quantitation limit of an individual analytical
procedure is the lowest amount of analyte in a sample
which can be quantitatively determined with suitable
precision and accuracy.

• The quantitation limit is a parameter of quantitative
assays for low levels of compounds in sample matrices,
and is used particularly for the determination of
impurities and/or degradation products.

• Several approaches for determining the quantitation
limit are possible, depending on whether the procedure
is a non-instrumental or instrumental.

65

Based on Visual Evaluation

• Visual evaluation may be used for non-
instrumental methods but may also be used with
instrumental methods.

• The quantitation limit is generally determined by
the analysis of samples with known
concentrations of analyte and by establishing the
minimum level at which the analyte can be
quantified with acceptable accuracy and
precision.

66

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 53

Based on
Signal-to-Noise Approach

• This approach can only be applied to analytical
procedures that exhibit baseline noise.

• Determination of the signal-to-noise ratio is
performed by comparing measured signals from
samples with known low concentrations of
analyte with those of blank samples and by
establishing the minimum concentration at which
the analyte can be reliably quantified.

• A typical signal-to-noise ratio is 10:1.

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Based on the Standard Deviation of the
Response and the Slope

• The quantitation limit (QL) may be expressed as:
QL = 10 /S
where  = the standard deviation of the response
S = the slope of the calibration curve

• The slope S may be estimated from the calibration
curve of the analyte.

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Estimate of 

• Based on Standard Deviation of the Blank

– Measurement of the magnitude of analytical background
response is performed by analyzing an appropriate number of
blank samples and calculating the standard deviation of these
responses.

• Based on the Calibration Curve

– A specific calibration curve should be studied using samples,
containing an analyte in the range of QL.

– The residual standard deviation of a regression line or the
standard deviation of y-intercepts of regression lines may be
used as the standard deviation.

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Recommended Data

• The quantitation limit and the method used for
determining the quantitation limit should be
presented.

• The limit should be subsequently validated by the
analysis of a suitable number of samples known
to be near or prepared at the quantitation limit.

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 55

LOD and LOQ : Estimated by

1. Based on Visual Evaluation
- used for non-instrumental methods

2. Based on Signal-to-Noise ratio
- 3:1 for LOD
- 10:1 for LOQ

3. Based on Standard Deviation of the Response and
the Slope

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Robustness

Small changes do not affect
the parameters of the assay

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ROBUSTNESS

• The robustness of an analytical procedure is a measure of its
capacity to remain unaffected by small, but deliberate variations
in method parameters and provides an indication of its reliability
during normal usage.

• The evaluation of robustness should be considered during the
development phase and depends on the type of procedure under
study.

• If measurements are susceptible to variations in analytical
conditions, the analytical conditions should be suitably controlled
or a precautionary statement should be included in the
procedure.

• One consequence of the evaluation of robustness should be that a
series of system suitability parameters (e.g., resolution test) is
established to ensure that the validity of the analytical procedure
is maintained whenever used.

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Typical Variations

• stability of analytical solutions,
• extraction time
Liquid chromatography:
• influence of variations of pH in a mobile phase,
• influence of variations in mobile phase composition,
• different columns (different lots and/or suppliers),
• temperature,
• flow rate.
Gas chromatography:
• different columns (different lots and/or suppliers),
• temperature,
• flow rate.

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 57

SYSTEM SUITABILITY TESTING

• System suitability testing is an integral part of many
analytical procedures.

• The tests are based on the concept that the equipment,
electronics, analytical operations and samples to be
analyzed constitute an integral system that can be
evaluated as such.

• System suitability test parameters to be established for a
particular procedure depend on the type of procedure
being validated. They are especially important in the case
of chromatographic methods.

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System Suitability in Chromatography

• To verify that the resolution and reproducibility of the
chromatographic system are adequate for the analysis to
be done

• The resolution, R, is specified to ensure that closely
eluting compounds are resolved from each other

• Replicate injections of a standard preparation are
compared to ascertain whether requirements for
precision are met

• The tailing factor, T, has to meet a certain requirement,
because as peak asymmetry increases, integration, and
hence precision, becomes less reliable

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Default Values for System Suitability for
HPLC Method

• Capacity factor
k' > 2

• Precision/Injection repeatability
• For %RSD < 2.0%, Five replicates
• For %RSD > 2.0%, Six replicates

• Resolution factor
Rs > 2 (Major peak and closest eluting)

• Tailing factor
T<2

• Theoretical Plates
In general N > 2000

77

Strategy for

Validation of Methods

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 59

Possible steps for a complete method validation:

1. Develop a validation protocol or operating
procedure for the validation

2. Define the application, purpose and scope of
the method

3. Define the performance parameters and
acceptance criteria

4. Define validation experiments
5. Verify relevant performance characteristics

or/and of equipment
6. Qualify materials eg. Standard and reagent
7. Perform pre-validation experiments

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Possible steps for a complete method validation:

8. Adjust method parameter acceptance criteria if
necessary

9. Perform full validation experiments
10. Develop SOPs for executing the method in

routine
11. Define criteria for revalidation
12. Define type and frequency of system suitability

tests and/or analytical control checks for the
routine
13. Document validation experiments and results in
validation report

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The validation protocol should include:

• Objective
• Scope
• Step by step procedure
• Method of evaluation
• Acceptance criteria
• Documentation and report
• Recommendation

81

Ex : Sum-up of the validation protocol (assay X: API)

Parameters Acceptance criteria

 Specificity - no peak interfering with
the peak of X
 Linearity
Correlation coefficient (r) - resolution nlt 1.5
y-intercept
> 0.995
 Accuracy < 2%
Recovery on a spiked placebo at 80%
Recovery on a spiked placebo at 100% 98-102%
Recovery on a spiked placebo at 120% 98-102%
98-102%
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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 61

Ex : Sum-up of the validation protocol (assay X: API)

Parameters Acceptance criteria

 Precision < 2%
Repeatability : RSD (n=6) % < 2%
Intermediate precision : RSD%
RSD < 2%
 Robustness Recovery : 98-102%
Check:
- Influence of modification of
pH of the mobile phase
- Influence of the composition
of the mobile phase
- Influence of the column

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Analyte conc vs. precision within and between days

Analyt % Analyte ratio Unit RSD (%)

100 1 100% 1.3
10 10-1 10% 1.8
1 10-2 1% 2.7
0.1 10-3 0.1% 3.7
0.01 10-4 100 ppm 5.3
0.001 10-5 10 ppm 7.3
0.0001 10-6 1 ppm 11
0.00001 10-7 100 ppb 15
0.000001 10-8 10 ppb 21
0.0000001 10-9 1 ppb 30

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Analyte recovery at different concentrations

Active Ingred. Analyte ratio Unit Mean recovery
(%) (%)
100%
100 1 10% 98-102
1% 98-102
> 10 10-1 0.1% 97-103
100 ppm 95-105
> 1 10-2 10 ppm 90-107
1 ppm 80-110
> 0.1 10-3 100 ppb 80-110
10 ppb 80-110
0.01 10-4 1 ppb 60-115
40-120
0.001 10-5

0.0001 10-6

0.00001 10-7

0.000001 10-8

0.0000001 10-9

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Validation report should be prepared that includes:

• objective and scope of the method (applicability, type)
• type of compounds and matrix
• detailed chemicals, reagents, reference standards and

chemicals used
• procedures for quality checks of standards and chemicals

used
• safety considerations
• method parameters
• critical parameters indicated from the robustness testing
• listing of equipment and its functional and performance

requirements, e.g. cell dimension, baseline noise, column
temperature range

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 63

Validation report should be prepared that includes:
• detailed conditions on how the experiments were

conducted, including sample preparation
• statistical procedures and representative calculations
• procedures for quality control in the routine (e.g.

system suitability tests)
• representative plots, e.g. chromatograms, spectra and

calibration curves
• method acceptance limit performance data
• the expected uncertainty of measurement results
• criteria for revalidation
• person who developed and initially validated the method
• summary and conclusion

87

Some Common Mistakes

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Some Common Mistakes

1. Validation range
- different from assay range
- not cover conc. range of content uniformity,

dissolution etc. (in case of same method)
- not cover conc. for impurity determination (compare
peak impurity with API peak)

89

Some Common Mistakes :

2. accuracy

- 9 determination VS. 9 injections
- different sample preparation

3. acceptance criteria

- no predetermined acceptance criteria in the
protocol

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 65

Some Common Mistakes :

4. stress test
- force degradation 10-20%
- each condition may be different
- show no interference

5. others
- stability indicating method?
- consistency of data and chromatograms

91

Conclusion of Method Validation

 One must consider the type of method and
product for analysis when choosing Validation
Parameters and Acceptance Criteria
– All results must be reported
– Compare data vs. pre-determined criteria
– Appropriate supervisory review
– Review and approval by QA for regulatory use

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Thank you

93

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 67

Promporn Jamnongtanachot

E-mail Address:
[email protected]

Telephone Number:+��� ���� ����
Education:
1998 – ���� Master of Sciences in Pharmacy (M.Sc. Pharm),

Chulalongkorn University
1991 – ���� Bachelor of Sciences in Pharmacy (B.Sc. Pharm),

Prince of Songkla University

Work Experience:
2007 – Present Quality Assurance Manager, Medica Innova Co., Ltd.

Invited lecturer, Faculty of Pharmaceutical Sciences, Prince
of Songkla University, Chulalongkorn University, Mahidol
University, Srinakharinwirot University, etc.
2002 – ���� Quality Control Manager, V. S. Pharma (����) Co., Ltd.
1996 – ���� Medical Scientist, Department of Medical Sciences, Ministry
of Public Health

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ภญ. แคทลียา นิรงั สรรค์

ตาํ แหน่งปัจจบุ นั
หวั หน้าแผนกพฒั นาวธิ วี เิ คราะห์ กองมาตรฐานการผลติ
ฝ่ายประกนั คณุ ภาพ องคก์ ารเภสชั กรรม
ประวตั ิการศึกษา
- เภสชั ศาสตรบ์ ณั ฑติ จฬุ าลงกรณ์มหาวทิ ยาลยั
- เภสชั ศาสตรม์ หาบณั ฑติ จุฬาลงกรณ์มหาวทิ ยาลยั
ประวตั ิการทาํ งาน
- เภสชั กรประจาํ แผนกมาตรฐานผลติ ภณั ฑ์ กองมาตรฐานผลติ ภณั ฑ์

ฝ่ายประกนั คณุ ภาพ องคก์ ารเภสชั กรรม (เมษายน 2541-กนั ยายน
2547)
- เภสชั กรประจาํ แผนกพฒั นาวธิ วี เิ คราะห์ กองมาตรฐานการผลติ ฝ่าย
ประกนั คณุ ภาพ องคก์ ารเภสชั กรรม (ตุลาคม 2548-กนั ยายน 2552)
- หวั หน้าแผนกพฒั นาวธิ วี เิ คราะห์ กองมาตรฐานการผลติ ฝ่ายประกนั
คุณภาพ องคก์ ารเภสชั กรรม (ตุลาคม 2552-ปัจจบุ นั )
- เป็นผตู้ รวจประเมนิ ทางวชิ าการ (Technical Assessor) ตามมาตรฐาน
มอก. 17043 (ISO/IEC 17043)ใหก้ บั สาํ นกั งานมาตรฐาน
ผลติ ภณั ฑอ์ ุตสาหกรรม (ตุลาคม 2558 - ปัจจุบนั )
- สอนเรอ�ื ง analytical method validation คณะเภสชั ศาสตร์
มหาวทิ ยาลยั บรู พา (2561)

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 69

Application of Best Practice in
Analytical Method Validation

(PART I)

Promporn Jamnongtanachot

12 November 2019

1

Validation Characteristics

Specificity Detection Limit (DL)

Linearity Quantitation Limit (QL)

Range Robustness

Accuracy Stability of Solution

Precision System Suitability

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Type of Analytical Procedure
 Identification
 Impurity test

• Quantitative test
• Limit test
 Assay
• Dissolution
• Content/Potency

3

System Suitability

4

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 71

System Suitability

Example of acceptance criteria
 Resolution: > 2
 Theoretical plate: > 2000
 Symmetry factor: 0.8 - 1.5
 %RSD:

- %RSD < 2, 5 replicate injections
- %RSD > 2, 6 replicate injections
 Signal-to-noise ratio: > 10 at reporting threshold

5

System Suitability

Example: Assay

Monograph
Reference solution (a): Standard solution
Reference solution (c): System suitability solution (containing impurities A, B and C)
System suitability:
- resolution: minimum 2.0 between the peaks due to API and impurity B, Reference
solution (c)

Acceptance criteria
 Resolution: NLT 2 between API and Impurity B, Reference solution (c)
 Symmetry factor: 0.8 - 1.5 for API, Reference solution (a)
 %RSD: NMT 2 for peak response of API, Reference solution (a)

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System Suitability

Reference solution (c)

More than 2

7

System Suitability

Reference solution (a) Peak Response Symmetry Factor
20195856 1.14
Injection No. 20246305 1.13
1 20388367 1.14
2 20357745 1.13
3 20334117 1.13
4 20304478 -
5 0.40 -

Average
%RSD

Less than 2 0.8 – 1.5

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 73

System Suitability

Example: Related substances test

Monograph
Reference solution (b): System suitability solution (containing API and impurities A,
D, G, K and L)
System suitability:
- peak-to-valley ratio: minimum 2.5, where Hp = height above the baseline of the 1st
of the 2 peaks due to impurity G and Hv = height above the baseline of the lowest
point of the curve separating this peak from the peak due to API epimer A (the 2nd of
the 2 principal peaks); and minimum 3, where Hp = height above the baseline of the
peak due to impurity L and Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to API epimer B (the 1st of the 2
principal peaks), Reference solution (b)

9

System Suitability

Epimer B Epimer A Reference solution (b)

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System Suitability

More than 3
More than 2.5

11

Specificity

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 75

Specificity

Assay (Content) Standard Internal std Sample
(as if)
Diluent Placebo

Assay (Dissolution)

Medium Placebo Standard Sample
in medium in medium in medium

13 Specificity Standard

Impurity Test Placebo

Diluent

Known impurity A Known impurity B Sample

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Specificity

Example: Diluent
analysis of B Tablets

Placebo

15

Specificity

Standard

Sample

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สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 77

Specificity

Example: Forced Degradation 10-20 % Degradation

Degradation Condition Duration

Type Store at room temperature 20 h
Control Expose to light (fluorescent lamp with intensity 96 h
Photolytic

Thermal NLT 12500 lux) 20 h
Hydrolysis Heat at 70 ºC 20 h
Acid hydrolysis Add water and heat at 70 ºC 20 h
Basic hydrolysis Add 0.1 N Hydrochloric acid and heat at 70 ºC 20 h
Oxidative Add 1 N Sodium hydroxide and heat at 70 ºC 20 h
Add 10 % Hydrogen peroxide and heat at

70 ºC

17

Specificity

Acid Hydrolysis

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Specificity

Oxidative

19

Linearity and Range

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Linearity and Range

Assay (Content)

Example: analysis of B Tablets
(Test concentration is 0.20 mg/mL)

% of Test concentration

50 % 75 % 100 % 125 % 150 %

0.10 mg/mL 0.15 mg/mL 0.20 mg/mL 0.25 mg/mL 0.30 mg/mL

21

Linearity and Range

Assay (Content)

Linearity and Range Residual plot

1000000 y = 31074219x + 20416 20000
800000 R² = 0.998
600000
400000 0.05 0.1 0.15 0.2 0.25 0.3 0.35
200000 Concentration (mg/mL)
Peak Response0 0
Residual0 0.000 0.010 0.020 0.030 0.040

-20000

Concentration (mg/mL)

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Linearity and Range

Assay (Dissolution)

Example: dissolution for C Tablets (controlled released product) cover a
region from 30%, after 1 h, up to 90%, after 24 h

10 – 110%

10 – 50% 70 – 110%

1 h (30%) 24 h (90%)

10 % 50 % 75 % 100 % 125 % 150 %

23

Linearity and Range

Impurity Test

Example: limit of each impurity is not more than 0.2 % and reporting
threshold is 0.05 %

Should be at least 0.05 % % of Sample solution
that is reporting threshold,
ICH Q3A and Q3B

QL 0.1 % 0.15 % 0.2 % 0.3 %

24

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 81

Linearity and Range

How to prepare 0.05 % ?

It is concentration of impurity in relative to concentration of active ingredient

in sample solution.

Example: analysis of imp. A in B Tablets

Sample solution is 1 mg/mL of B 0.05 % of Sample
Standard solution is 2 µg/mL of Imp. A (0.2 %) solution

5.0 mL

Mobile phase

Standard solution 20-mL VF
(2 µg/mL of Imp. A) (0.5 µg/mL of Imp. A)

25

Accuracy

26

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82 TIPA | Thai Industrial Pharmacist Association

Accuracy

Assay (Content)

Example:
Analysis of B Tablets by HPLC (Test concentration is 0.20 mg/mL)
Procedure:
Prepare standard solution and 9 simulated samples

Standard solution
B standard 20 mg

100-mL VF

27

Sim. sample 50 % Accuracy Sim. sample 150 %
B standard 10 mg B standard 30 mg
Placebo 0.4981 g Sim. sample 100 % Placebo 0.4981 g
B standard 20 mg
Placebo 0.4981 g

100-mL VF (50-1) 100-mL VF (100-1) 100-mL VF (150-1)

100-mL VF (50-2) 100-mL VF (100-2) 100-mL VF (150-2)

100-mL VF (50-3) 100-mL VF (100-3) 100-mL VF (150-3)
28

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 83

Accuracy

Sample Added Found %Recovery Mean of
Amount (mg) % Recovery
50-1 Amount (mg) 101.4
50-2 10.23 10.37 100.7 101.1
50-3 10.07 10.14 101.2
100-1 9.91 10.03 101.2 100.6
100-2 19.98 20.22 100.3
100-3 20.11 20.17 100.0 99.8
150-1 20.08 20.08 100.7
150-2 30.14 30.35 99.5
150-3 30.06 29.91 99.3
29.99 29.78

29

Accuracy

Assay (Dissolution)

Example: Dissolution for C Tablets (500 mg)
• Apparatus II, 100 rpm
• Medium: Phosphate buffer pH 6.8, 1000 mL
• Tolerance: 30 – 40 %LA after 1 h and NLT 90 %LA after 24 h
Procedure:
Prepare standard solution and 12 simulated samples

Standard solution
C standard 50 mg

100-mL VF

30

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84 TIPA | Thai Industrial Pharmacist Association

Accuracy

Sample 10 % Sample 50 % Sample 100 % Sample 150 %
C std 5 mg C std 25 mg C std 50 mg C std 75 mg
Placebo 0.3 g Placebo 0.3 g Placebo 0.3 g
Placebo 0.3 g in Medium in Medium in Medium
in Medium

100-mL VF (10-1) 100-mL VF (50-1) 100-mL VF (100-1) 100-mL VF (150-1)

100-mL VF (10-2) 100-mL VF (50-2) 100-mL VF (100-2) 100-mL VF (150-2)

100-mL VF (10-3) 100-mL VF (50-3) 100-mL VF (100-3) 100-mL VF (150-3)
31

Accuracy

Impurity Test

Example:
Analysis of Impurity A in B Tablets (limit of Impurity A is not more than 0.2 %)
Procedure:
• Prepare standard solution
• Prepare 3 non-spiked samples
• Prepare 3 spiked samples at 0.05 %
• Prepare 3 spiked samples at 0.2 %
• Prepare 3 spiked samples at 0.3 %

32

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 85

Accuracy

Non-Spiked

• 1 g of sample powder (eq. to 100 mg of B)

1 mg/mL of B

Spiked sample at 0.05 % 100-mL VF

• 1 g of sample powder (eq. to 100 mg of B) 1 mg/mL of B
• 1 mL of Impurity A stock solution (0.05 mg/mL) 0.0005 mg/mL of Impurity A

100-mL VF
33

Accuracy 1 mg/mL of B
0.002 mg/mL of Impurity A
Spiked sample at 0.2 %

• 1 g of sample powder (eq. to 100 mg of B)
• 1 mL of Impurity A stock solution (0.2 mg/mL)

Spiked sample at 0.3 % 100-mL VF

• 1 g of sample powder (eq. to 100 mg of B) 1 mg/mL of B
• 1 mL of Impurity A stock solution (0.3 mg/mL) 0.003 mg/mL of Impurity A

100-mL VF

34

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86 TIPA | Thai Industrial Pharmacist Association

Precision

35

Precision

Assay (Content)

Example:
Analysis of B Tablets by HPLC (Test concentration is 0.20 mg/mL)

Standard solution Repeatability
B standard 20 mg
Sample solution
100-mL VF 0.5 g of sample powder

(eq. to 20 mg of B)

Homogeneous sample

100-mL VF

36

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 87

Precision

Sample Exp. 1 % LA Exp. 3
98.4 Exp. 2 98.8
1 99.7 98.6 99.3
2 99.8 100.7 99.2
3 99.0 99.5 98.8
4 99.8 99.1 99.8
5 99.9 98.2 99.2
6 99.4 98.3 99.2
Mean (n=6) 0.6 99.1 0.4
% RSD (n=6)
% RSD (n=18) 0.9
0.7
37

Precision

Assay (Dissolution)

Example:
Analysis of B Tablets by HPLC (Each B Tablets contains 20 mg of B)

Standard solution Repeatability
B standard 20 mg in Medium
Sample solution
100-mL VF 0.5 g of sample powder
(eq. to 20 mg of B) in Medium

Homogeneous sample 100-mL VF

38

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88 TIPA | Thai Industrial Pharmacist Association

Precision

Standard solution Intermediate precision
B standard 20 mg in Medium

100-mL VF Mean of % Dissolved
39
Compare mean of % Dissolved between
experiments
Acceptance Criteria
• < 85 % Dissolved, differ NMT 10 % absolute
• > 85 % Dissolved, differ NMT 5 % absolute

Same lot

Detection Limit
and

Quantitation Limit

40

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 89

Detection Limit and Quantitation Limit

Impurity Test

Example: limit of each impurity is not more than 0.2 % and reporting
threshold is 0.05 %

0.0005 mg/mL of QL concentration DL concentration
Impurity A
(0.05 %)

Prepare A standard at Dilute the solution Dilute the solution
reporting threshold and unit signal-to-noise unit signal-to-noise
measure signal-to-noise ratio is about 10:1 ratio is about 3:1

ratio

41

Detection Limit

Prepare spiked placebo at Detection limit

Spiked placebo at DL concentration

• 0.9 g of placebo
• 1 mL of Impurity A stock solution (xx mg/mL)

9 mg/mL of Placebo
Impurity A at DL concentration

Signal-to-noise ratio is about 3:1

100-mL VF

42

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90 TIPA | Thai Industrial Pharmacist Association

Quantitaion Limit

Prepare spiked samples for Accuracy and Precision at Quantitation limit

Standard solution Spiked sample at QL
concentration
Impurity A at QL
concentration • 1 g of sample powder (eq. to 100
mg of B)
100-mL VF • 1 mL of Impurity A stock solution
(xx mg/mL)

Non-Spiked sample 1 mg/mL of B
Impurity A at QL concentration
• 1 g of sample powder (eq. to
100 mg of B)

1 mg/mL of B

100-mL VF 100-mL VF
43

Robustness

44

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 91

Robustness

Assay (Content)

Example:

Analysis of B Tablets by HPLC

System suitability requirement:

• Symmetry factor: 0.8 - 1.5 for B, Standard solution

• %RSD: NMT 2 for peak response of B, Standard solution

Mobile phase: Vary ratio of mobile phase
Buffer pH 3.0 : ACN (45 : 55) • Buffer pH 3.0 : ACN (35 : 65)
• Buffer pH 3.0 : ACN (55 : 45)

Vary pH of buffer
• Buffer pH 2.8
• Buffer pH 3.2

45

Robustness

Injection No. Buffer pH 2.8 Buffer pH 3.2

1 Response Symm. Fac. Response Symm. Fac.
2
3 654740 0.98 651052 0.98
4
5 652005 0.98 651132 0.98
Mean
% RSD 645878 0.98 650386 0.98

650031 0.98 651833 0.98

649937 0.98 651337 0.98

650518 - 651148 -

0.5 - 0.1 -

46

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92 TIPA | Thai Industrial Pharmacist Association

Robustness

Buffer pH 3.0 : ACN Buffer pH 3.0 : ACN

Injection No. (35 : 65) (55 : 45)
Response Symm. Fac. Response Symm. Fac.
1
2 663627 1.13 655535 0.93
3
4 663841 1.14 655455 0.93
5
Mean 659928 1.14 653856 0.94
% RSD
658973 1.14 653245 0.94

663492 1.13 655398 0.94

661972 - 654698 -

0.4 - 0.2 -

47

Stability of Solutions

48

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 93

Stability of Solution

Assay (Content)

Example:
Analysis of B Tablets by HPLC (Test concentration is 0.20 mg/mL)
Procedure:
Prepare standard solution and sample solution

Standard solution Sample solution
0.20 mg/mL 0.20 mg/mL

Kept in autosampler

49 Stability of Solution tn

t0 t1 Freshly
prepared std
Freshly
prepared std

Calculate concentration at each time point using a freshly prepared standard
and calculate % Remaining by compare with the initial analysis
Acceptance Criteria
% Remaining should be between 98 – 102

50

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51

สมาคมเภสชั กรอุตสาหการ (ประเทศไทย) 95

Application of Best Practice in
Analytical Method Validation

(Part II)

Katthaleeya Nirungsan
Nov. 12, 2019

1

Example: BP 2019

2

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96 TIPA | Thai Industrial Pharmacist Association

3
4